PRACTICAL WORKBOOK IN HISTOLOGY, CYTOLOGY AND EMBRYOLOGY - 2016

Chapter 1. HISTOLOGICAL TECHNIQUE.

General guidelines for studying histological slides

Mastering the subject of Histology, Cytology, and Embryology involves acquiring knowledge of the microscopic and submicroscopic Structure of human Cells, Tissues, and Organs using microscopic instruments. During Practical Classes, students work with a Light Microscope and examine histological or embryological specimens.

The examination of a specimen begins with a preliminary Overview. Before placing it on the microscope stage, it should be inspected with the naked eye. This allows one to see, first of all, where the coverslip is located, and second, if there is no label, such an inspection often helps to deduce from which tissue the section was made.

One can also examine the specimen using only the eyepiece. To do this, remove the eyepiece from the tube and use it as a low-power magnifying Glass. Take the specimen in your left hand, hold it vertically near a light source, and with your right hand bring the upper lens of the eyepiece closer to the specimen while looking through the lower lens. By moving the eyepiece closer to or further from the specimen, a sharp image can be obtained, allowing its overall structure to be examined and studied.

Next, place the microslide on the microscope stage and examine it under low magnification. Low magnification provides a view of a fairly large area of the section (with an 8x objective and a 10x eyepiece, the diameter of the field of view is approximately 1200 µm). By moving the specimen, one can explore the entire surface of the section. Such an examination makes it possible to determine from which organ or tissue the section was made and to locate the areas most suitable for a deep, thorough study of histological structures. Image clarity in different PARTS OF THE specimen depends on the plane of the section and the quality of specimen Processing.

When moving the specimen, one should keep in mind that the microscope produces an inverted image; that is, moving the specimen from top to bottom will cause the image to move from bottom to top, and moving it from left to right will cause the image to shift from right to left.

High magnification of the microscope is required for a detailed study of a specific area of the specimen. Compared to low magnification, with a 40x objective and a 10x eyepiece, the diameter of the field of view decreases to 375 µm. Therefore, to ensure this area falls within the microscope's field of view, make sure it is positioned in the center of the field under low magnification before switching from low to high power. It is very important to remember that the fine adjustment knob (micrometer screw) must be used carefully, turning it slowly in both directions — clockwise and counterclockwise. Examining the specimen at high magnification also makes it possible to study the specimen in depth across various focal planes. If high magnification fails to bring the objective into focus, this may be due to the specimen being placed incorrectly, i.e., with the coverslip facing downward.

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Fig. 4. Diagram illustrating how the perception of an object's shape and structure changes depending on the plane of section.

A, B — transverse sections; C, D — longitudinal sections

Fig. 5. Diagram showing the appearance of sections made in different planes through an object divided by partitions (an orange):

A — transverse section, B — oblique section, C — longitudinal section.

Fig. 6. Diagram showing the appearance of straight tubes in cross-sections when sections are made in different planes.

A — transverse sections, B — oblique sections, C — longitudinal sections.

Fig. 7. Diagram showing the appearance of sections through a bent tube.

Fig. 8. Diagram showing the appearance of sections through a cable-like structure.

A — transverse section, B — oblique section, C — longitudinal section.

When working with a microscope, you should learn to look with both eyes without squinting. Squinting is harmful to THE EYE AND causes it to tire quickly. Therefore, you should immediately train yourself to look into the eyepiece with your left eye while keeping your right eye open.

When attempting to interpret what is visible on the specimen, certain difficulties arise. It must be remembered that a specimen is merely a single section, based on which we form an impression of The structure of the tissue or organ as a whole. The cells we examine consist of two main parts — The Nucleus and the Cytoplasm. In stained specimens, the nucleus is always darker in color with clear, rounded contours, bounded by the nuclear envelope or membrane. One or more intensely stained nucleoli and small clumps of Chromatin are often observed within the nucleus. The cytoplasm surrounds the nucleus. Using a light microscope, Organelles and inclusions in the cytoplasm can be detected only by applying special staining techniques. The Cell membrane is not visible.

Despite having different shapes, cells are three-dimensional structures. The shape of a cell in a section depends on its overall Morphology, on the one hand, and the plane of the section, on the other. Fig. 4 schematically illustrates how our perception of the shape and structure of an object under study changes depending on the section plane. For example, let us take a hard-boiled egg and cut it. Cross-sections (A, B) and longitudinal sections (C, D) will look different depending on whether the cut passes through the yolk or not. Imagine that the yolk represents the Cell Nucleus, and the white represents its cytoplasm. In histological sections 5–7 µm thick, we observe cells larger than 15 µm in various ways. The nucleus (the yolk in the diagram) is not visible in every cell. When the nucleus falls within the section, it often appears smaller than it actually is (Fig. 4, D). The apparent size of cells in a section may also appear smaller, depending on the plane and the specific part of the cell through which the section passes (Fig. 4, A, C). Therefore, in sections, cells of similar structure and approximately equal volume can vary greatly in size.

In preparations of organs containing partitions or septa, histological structures also appear differently depending on how the section is made (Fig. 5) — transversely (A), obliquely (B), or longitudinally (C).

In addition to cells, various types and sizes of tubular structures are observed within organs. Blood and Lymphatic vessels, as well as Organs of the digestive, respiratory, and urogenital systems, have a tubular shape. Straight tubes (Fig. 6) can be cut transversely (A), obliquely (B), and longitudinally (C), thus taking on different forms in the preparation.

Bent tubes in sections cut in different planes look approximately as shown in Fig. 7. When studying organs, one may encounter structures whose sections resemble a cable cross-section (Fig. 8). These are transverse (A), longitudinal (C), and oblique (B) sections of nerve trunks.

When examining a histological preparation, one may encounter artifacts. In histology, an artifact refers to features or structures that appear in a preparation accidentally or due to improper handling of organ fragments or their sections. Distortion of the true appearance of tissue can occur at any stage of preparation manufacturing. The artifacts most commonly found in preparations are described below.

1. Post-mortem tissue decay phenomena due to delayed or inadequate tissue fixation. In such preparations, many cellular structures cannot be identified.

2. Cell shrinkage caused by a sharp change in the concentration of fixative solutions or embedding tissue fragments in overly hot paraffin. This leads to The formation of gaps and cracks in the preparations, as different tissue components may pull apart from one another.

3. Precipitation of sediments resulting from errors in the chemical processing of tissue pieces and sections. Isolated or clustered deposits of colored particles and clumps appear in the preparations. For example, formalin pigment appears as a brown granular precipitate resulting from the interaction of an acidic formalin solution with erythrocyte Hemoglobin or Muscle tissue Myoglobin.

4. Folds and wrinkles occur when thin paraffin sections are transferred onto a glass microscope slide. After staining, they appear in the preparation as heavily stained areas with a high density of cells.

5. Nicks and irregularities on the microtome knife leave long, straight scratches on the preparation with disrupted histological structure.

6. Small air bubbles form when a coverslip is improperly placed over the section, appearing in the preparation as transparent, colorless globules.

7. Occasionally, foreign impurities introduced during preparation and the handling of various Materials are observed in the specimen, such as cotton, wool, and silk fibers.

The Study of a histological preparation is traditionally accompanied by making a drawing. This drawing records the main features and structural peculiarities of a given organ or tissue. Sketching helps to better understand the micropreparation, learn to identify its primary structures, and pay attention to structural details. A drawing AIDS in memorizing the preparation and fosters a deeper comprehension of the theoretical material.

To learn how to recognize Cells and Tissues of organs in a histological preparation, it is helpful to compare what is seen under the microscope with illustrations in atlases, textbooks, practical manuals, and guidebooks accompanied by explanatory labels. Since most illustrations (drawings, microphotographs) are black-and-white while micropreparations are stained, one should keep in mind that the degree of staining generally corresponds to the intensity of nuclear staining with hematoxylin, whereas all shades of gray reflect the degree of cytoplasmic staining with eosin.

For sketching preparations during practical classes, students must have a drawing album and colored pencils. It is advisable to use a wide range of colored pencils in various tones to accurately render the coloration of different structures from the micropreparations in the drawings. Drawing with colored inks or felt-tip pens is not permitted.

A drawing must reflect the main Structural Features of the organ or tissue, while structural details can be presented separately. Different parts of the preparation must be drawn in correct proportion to one another. Labels for the drawing should preferably be placed beneath it, using arrows and numbers to indicate their exact locations on the drawing. Each drawing must be accompanied by a title indicating the type of tissue or name of the organ, the staining method, and the magnification at which the preparation was examined.

In addition to sketching, students must learn to describe a micropreparation — that is, compile a study protocol of the object using specific "histological language," which constitutes the histological nomenclature (Histological Terminology: International Terms of Human Cytology and Histology / Ed. by Yu. B. Chaikovsky, O. D. Lutsyk. Kyiv: Medytsyna, 2010. 284 p.). This is a systematic list of concepts and terms that morphologists worldwide have agreed to use to designate specific microscopic structures or related processes. The protocol can be based on the micropreparation description provided in this practical guide. Next, one should present a comparison (Differential Diagnosis) with other organs similar to the one being studied and finally conclude whether the section belongs to a particular organ or tissue based on specific evidence.